The anaerobic organism Clostridium difficile is the major causative agent of antibiotic-associated bacterial diarrhea and pseudomembranous colitis (PMC) among mainly elderly patients in hospitals and long term care facilities [1,2]. The organism cannot compete successfully with the normal microbial flora in the adult colon, but when the normal intestinal microflora is altered, for example by antibiotic treatment, C. difficile is able to colonize the gut in high numbers. Antibiotic therapy accounts for 98% of all cases of C. difficile associated diarrhea (CDAD). However, any predisposing condition which alters the normal intestinal flora, including any condition which requires extensive immunosuppressive treatment, can also lead to the development of CDAD. For example, recent evidence suggests that AIDS patients are also high risk candidates for acquiring CDAD [3,4].
C. difficile produces two exotoxins, toxin A (an enterotoxin) and toxin B (a cytotoxin) which appear to play important roles in causing CDAD. Toxin A is primarily responsible for the disease. It acts by binding to epithelial cells in the intestine, resulting in the destruction of these cells and causing the secretion of fluid into the intestine. The destruction of these protective epithelial cells by toxin A represents the crucial step leading to the development of diarrhea. Once damage has occurred to the epithelial cells, the potent cytotoxin B can then gain access to underlying sensitive tissues and initiate additional clinical symptoms.
Toxin A has been found to display a lectin-like activity which allows it to bind to an oligosaccharide receptor on epithelial cells. Several oligosaccharide sequences have been identified as potential receptors for toxin A, and include the following structures [5-7]:
______________________________________ .alpha.Gal(1-3).beta.Gal(1-4).beta.GlcNac ______________________________________ .beta.Gal(1-4).beta.GlcNAc (human blood group antigen X) (1-3) .alpha.Fuc .beta.Gal(1-4).beta.GlcNAc (human blood group antigen Y) (1-2) (1-3) .alpha.Fuc .alpha.Fuc .beta.Gal(1-4).beta.GlcNac (human blood group antigen I) (1-6) .beta.Gal (1-3) .beta.Gal(1-4).beta.GlcNAc ______________________________________
In addition, highly purified toxin A preparations have been obtained using bovine thyroglobulin affinity columns which have terminal .alpha.Gal(1-3).beta.Gal(1-4).beta.GlcNAc oligosaccharide sequences [8,9].
The current therapy for patients who suffer from CDAD or PMC is to remove the offending drug and begin oral administration of the antibiotics Metronidazole or Vancomycin along with fluid replacement [3,14]. Vancomycin is only used in certain situations when patients cannot tolerate or are not responsive to Metronidazole treatment. In addition, Vancomycin is not used routinely because of its high cost. This form of therapy is effective in about 80% of the patients who suffer from CDAD or PMC. In about 20% of patients, the diarrhea returns after discontinuing antibiotic treatment [15]. In such individuals, episodes continue to recur until the normal intestinal flora is reestablished and the numbers of C. difficile organisms are reduced. This is a slow process, since antibiotics such as Metronidazole, which disturb the balance of the normal intestinal flora, are administered each time the diarrhea occurs.
The only other treatment for CDAD and PMC which removes toxin activity from the intestinal tract involves the use of multigram quantities of anion exchange resins such as cholestyramine and colestipol given orally in combination with antibiotics. This approach has been used to treat mild to moderately ill patients, as well as individuals who suffer from multiple episodes of diarrhea [16,17]. This form of therapy has achieved only moderate success in treatment of the disease [18]. In addition to the lack of efficacy of ion exchange resins, there are several other disadvantages associated with the use of resins. Ion exchange resins do not bind specifically to toxin A. Thus, they may bind to antibiotics themselves, resulting in suboptimal levels of antibiotic within the gut. In addition, if patients are receiving other medications that bind to ion exchange resins, there can be reduced drug levels. A further disadvantage of ion exchange resins is the disagreeable taste and aftertaste which are associated with oral administration of these compounds.
With respect to methods of diagnosis of the presence of toxin A in a sample, one method for detecting C. difficile in a sample is to culture the sample. The disadvantages of this method include the length of time required and interference by non-pathogenic, i.e. non-toxin producing, C. difficile strains. Other methods involve the use of specific antisera or monoclonal antibodies. U.S. Pat. Nos. 4,863,852 and 5,098,826 describe methods for detecting C. difficile toxin A by the use of reagents containing biological receptors for toxin A, including the .alpha.Gal(1-3).beta.Gal(1-4).beta.GlcNAc, X and Y antigen oligosaccharide sequences, bound to a support.
In view of the above, there is a need for a compound which would treat antibiotic associated diarrhea. A preferred compound would be administered noninvasively, such as orally.